CA2060864A1 - Non-a, non-b hepatitis related nucleic acids, agents antibodies and their detection reagents - Google Patents

Abstract

Abstract Oligopeptide spGORb-epi from non-A, non-B hepatitis (NANB) virus; detection reagents for antibody associated with NANB hepatitis using the aforementioned oligopeptide or fusion protein of such oligopeptide and conventional proteins;
monoclonal and polyclonal antibodies specific NANB hepatitis obtained by immunization of animals with the aforementioned oligopeptide as antigen; and detection reagents of NANB
hepatitis related antigens using the aforementioned specific antibodies.

Description

NON-A, NON-B HEPATITIS RELAT~D NUCLEIC ACIDS, ANTIG~NS, ANTIBODIES AND THEIR DETECTION REAGENTS

Introduction to the Invention The present invention relates to novel antigens, antibodies and non-A, non-B hepatitis diagnostic reagents using those materials.
The causative a~ent of non-A, non-B hepatitis (hereinafter NANB hepatitis) afflicts human beings with such diseases as acute hepatitis, fulminant hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. It presents itself as the major cause of post transfusion hepatitis in approximately 10~ of the blood recipients in Japan. The causative viruses of hepatitis A and hepatitis B
have been isolated and these diseases are under medical control. However, the causative agent of NANB hepatitis is a mystery although its presence was assumed over 10 years ago.
In 1988, a research group at Chiron Corporation in the United States announced that they had succeeded in cloning the gene of the causative agent, and introduced an immunoassay kit for detection of viral antibody speci~ic to NANB
hepatitis. That diagnostic reagent is now being evaluated at various hospitals, research institutions and blood centers.
According to Chiron's research group, the causative agent is a Flavivirus because of its genomic structure, and they have named it hepatitis C virus (HCV). The data reported to date have confirmed a good correlation between HCV antibody detected by their immunoassay kit and the course of the disease of NANB hepatitis. With respect to the HCV antibody, however, there are problems yet to be solved and elucidated:
its insufficient specificity and sensitivity (revealed in several reports), cross reaction between the HCV and a large part of auto-i~mune hepatitis and hepatitis B, and proof that what they call HCV is the real causative a~ent of NANB
hepatitis.
Non-A, non-B hepatitis specific diagnostic assays based Oll detection systems using the present invention in this and preceding applications (Japanese Patent Application Nos.
028191/90 filed February 9, 1990, 15388'7/90 filed June 14, 1990, and 335806/90 filed November 30, 1990) are capable of detecting patients not detected by the kit using Chiron's antigen ~Ortho HCV Ab ELISA test: Ortho Diagnostic Systems, 'rokyo, Japan).

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SUMMARY OF THE INVENTION
An object of the present invention is to provide previously unidentified antigens and antibodies required for detection of NANB hepatitis.
Nucleic acids and proteins disclosed in the present invention have usefulness in the diagnosis of NANB hepatitis and have been provided only by this invention as totally new materials.
The present invention discloses GOR gab DNA having the sequence as shown in list l; GOR gab protein having the sequence as shown in list 2; GOR47-1 RNA having the sequence as shown in list 3; GOR 47-1 RNAC having the sequence as shown in list 4; GOR47-1 DNA having the sequence as shown in list 5;
GOR47-1 DNAC having the sequence as shown in list 6; GOR47-1 Protein having the sequence as shown in list 7; oligopeptide spGORl having the sequence as shown in list 8; oligopeptide spGOR2 having the sequence as shown in list 9; oligopeptide spGORa-epi having the sequence as shown in list 10;
oligopeptide spGORb-epi having the sequence as shown in list 11; detection assay for nucleotides related to NANB hepatitis using such nucleotides as probe; detection assay for nucleotides related NANB hepatitis using such nucleotides as primer; detection assay for antibodies related to NANB
hepatitis using such proteins or peptides as antigen;
detection assay for antibodies related to NANB hepatitis using 2 ~

fusion protein with such proteins or peptides as antigen;
monoclonal or polyclonal antibodies obtained by immunization of animals with such proteins or peptides; and detection assay for NANB hepatitis related antigen using such monoclonal and polyclonal antibodies. These detection assays can be used as e~icient tools for diagnosis of NANB hepatitis.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the base sequence of recombinant lambda-gtll phage DNA to assemble GOR-47 DNA in EcoRI
cleavage. Sequence within the frame is GOR4'7-1 DNA and those on the left and right sides of the frame are lambda-gtl:L DNA.
Figure 2 shows reactivity the partial peptide on the ORF of GOR47-1 determined by EIA. Samples with O are NANB
hepatitis and those with O are hepatitis B derived; ~Gal~GOR
is fusion protein of ~-galactosidase and GOR~7-1; spGOR3 is the peptide extended by 26 residues from the 15th residue of spGORa-epi toward C terminus and having the sequence LPVPRNPCRGPSGLSPSLCPSQTSVL.
Figure 3 shows Gl and G2 used as primers for PCR.
Figure 4 is a histogram showing GORa antibody in samples from normal subjects and chronic hepatitis patients detected by~EIA using spGOR2 as antigen. On the ordinate is samples from normal subjects and chronic hepatitis patients detected by EIA using spGOR2 as antigen. On the ordinate is ~ 0 ~ L~

shown frequency. Absorbance 0.4 at ~92 is tentatively set as the cut-off value and shown by a dotted line. Positive rate for each group is as follows: (l) Normal subjects-1%; (2) NANB chronic hepatitis patients-'16%; (3) Chronic hepatitis B
patients-2%; (4) NANB liver cirrhosis-56%; (5) Hepatitis B
liver cirrhosis-7~; (6) NANB hepatoma-56%; (7) Hepatitis B
hepatoma-0%; (8) Lupoid hepatitis-0~; (9) Primary biliary cirrhosis-0~.
Figure 5 shows change with time of GORa antibody in acute NANB hepatitis patients determined by EIA using spGOR2.
O : IgG class GORa antibody; : IgM class GORa antibody;
O : HCV by Ortho HCV ELISA Ab test kit; black area: Serum ALT (alanine aminotransferase) value.
Figure 6 is distribution pattern of chronic NANB
hepatitis patient sera showing positive for GORa antibody.
Sera positive for Chiron's HCV antibody are shown by * mark.
Figure 7, a histogram, shows comparison of antibody to GORb epitope detection frequency using spGORb-epi in three groups: l) normal subjects, 2) type-B liver diseases and 3) NANB hepatitis. On the ordinate is samples from normal subjects and chronic hepatitis patients detected by EIA using spGORb-epi as antigen. On the ordinate is shown frequency.
Absorbance 0.4 at OD~92 is tentatively set as the cut-off value and shown by a dotted line.

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Figure 8 shows agarose gel electrophoresis patterns of the material produced by PCR.

DETAILED DESCRIPTION OF THE INVENTION

One feature of the present invention resides in the following:
Oligopeptide spGORb-epi (list 11) having the following amino acid sequence:
VAKQHVRDGR KDSLDGFV (Y) 18 amino acid residues correspond to amino acid numbers 346-363 of GOR gab protein, while C-terminal (Y) was added for peptide design. Generally (Y) is tyrosine for radiolabelling, though another amino acidts) can be utilized (e.g. cysteine) for combining with such a protein as alkaline phosphatase.
A, G, C, and U stand for Adenine, Guanine, Cytosine and Uracil respectively in the ribonucleic acids of this invention.
A, G, C, and T stand for Adenine, Guanine, Cytosine and Thymine respectively in the deoxyribonucleic acids of this invention.
C, P, R, K, A, E, T, G, V, D, Q, S, N, L, stand for Cysteine, Proline, Arginine, Lysine, Alanine, Glutamic acid, Threonine, Glycine, Valine, Aspartic acid, Glutamine, Serine, Asparagine and Leucine respectively in the proteins of this invention.

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The followin~ procedures were utilized:
1~ PreDarat on of animal models for NANB hepatitis Research started with preparation of animal models for NANB hepatitis. Various animals were injected with sera from donors which were known to have caused post transfusional NANB hepatitis in man. Among those animals, only chimpanzees responded and expressed symptoms similar to human NANB
hepatitis. In case of the experimental NANB hepatitis in chimpanzees, some characteristic ultrastructural changes (e.g., convoluted curved membrane, tubular structure, sponge-like structure, and microtubular aggregates) tend to appear in the cytoplasm of liver cells before clinical symptoms, thus making these ultrastructural changes a reliable marker;
experimental passage of NANB causative agent was undertaken from a human being to a chimpanzee, then from that chimpanzee to other chimpanzees.

2) Material for isolation of NANB he~atitis related aene Human serum (No. 30017) was injected into a chimpanzee (C3'7) and the serum ~rom that chimpanzee (in acute phase hepatitis) was injected into 5 chimpanzees (C41, C43, C45, C46 and C47). Among those 5 chimpanzees, plasma were taken from those in the phase positive for the aforementioned ultrastructural changes and pooled for injection into other 2 ~ 4 animals including the chimpanzee CHl9. Plasma taken from CHl9 ~in acute phase of NANB hepatitis) was used as the material for isolation of NANB hepatitis related gene.

3~ Extract n of nucleic acids form chim~anzee Plasma About 6 liters of plasma from the chimpanzee CHl9 (in acute phase o~ NANB hepatitis) was centrifuged on the J6 rotor (Beckman) at 3,000 rpm for 30 minutes, and the resulting supernatant was centrifuged on the Ti-15 rotor (Beckman) at 4C at 30,000 rpm for 5.3 hours. The resulting precipitate was suspended in 120 ml of 50 mM Tris-Cl (pH~/.5)/5mM EDTA to obtain virus-rich fraction concentrated by approximately 50 times. After addition to 6 ml of this fraction (equivalent to 300 ml of the original plasma) of 6 ml of SDS/Proteinase K
cocktail (400mM NaCl/20mM EDTA/4% SDS/lOOmM Tris-Cl buffer (pH
8.0)/Proteinase K 2 mg/ml) and overnight incubation at 3'/C, nucleic acids were extracted with phenol and precipitated by ethanol.

4) cDNA svnthesis Using 1/4 in volume (equivalent to 75 ml of the original plasma) of the nucleic acids obtained under 3) above, cDNA was ~ynthesized. After incubation of the template RNA at 65C for 3 minutes, primary cDNA strand was synthesized in two tubes, one tube with oligo-dT12, and the other with randam-hexanucleotide as primer. This series of cDNA synthesis, including synthesis of the secondary cDNA strand and blunting of double-stranded DNA termini, was conducted according to Gubler's method using a cDNA kit (Amersham, U.K.) 5) PreDaration of _DNA librarv After protecting possible EcoRI cleavage sites in the double-stranded cDNA prepared in 4) above by EcoRI
methylase, providing both ends with EcoRI linker, it was integrated into lambda-gtll DNA at the EcoRI site and assembled with phage protein to make recombinant phage.
Series of this reaction was processed using the lambda-gtll cloning kit (Amersham, U.K.). Library size of cDNA primed by oligo dT12 and that of cDNA primed by randam-hexanucleotide were 1.0 x 106 PFU and 4.3 x 106 PFU respectively.

6) Screeninq of cDNA librarY
cDNA libraries prepared under ~) were searched for a NANB hepatitis related cDNA clone by immunoscreening using two antibodies. After infecting E.coli Y1090 with the above recombinant phage, and dispensing it on LB-Agar plates for incubation at 43C for 3 hours, a filter impregnated with IPTG
was placed on it and incubated at 37C for 3 hours. The filter was then removed and washed with the buffer and primary antibody was added to it. A mixture of plasma of a chimpanzee _ g 2 ~

infected with NAN~ hepatitis, a human plasma known to have caused NANB hepatitis by needle stick accident, and a plasma of chronic NANB hepatitis patient were used as the primary antibody, and incubated at 4C overnight. After washing with the buffer and addition of the secondary antibody tperoxidase labeled antihuman I~G), it was incubated at room temperature for 30 minutes. It was then washed with the buffer and added with the mixture of DAB ~3,3'-Diaminobenzidine tetrahydrochloride; Sigma, USA), Ni, Co, HaO2 for color development. If there were fractions of NANB related genes in the cDNA libraries, and if they fused with the open reading ~rame (ORF) of ~-galactosidase of lambda-gtll phage DNA in-frame, there should be fusion protein expressed by E coli infected with the phage and, upon its recognition by NANB
related antibody, it would bind to the antibody to give a positive signal. Inventors' experiment con~irmed that point.
Among several clones giving positive signals in the screening test, there was a clone named GOR4'7-1 described hereafter.

7) Determination of the nucleotide seauence of NANB
heDatitis related cDNA clone (GOR47-1) The nucleotide sequence of GOR47-1 obtained under 6) above was determined by Sanger's method by puri~ying it, cleaving its DNA by ~coRI to take out cDNA, and subcloning it to EcoRI site of Phagescript (trade name of Stratagene, USA~.
By means of primer derived from DNA phage, the nucleotide sequence at the insertion site of GOR~-7-l was simultaneously determined in the same m0thod. As a result, it was confirmed that GOR47-l DNA was inserted into lambda-gtll DNA by the sequence and direction shown in Figure l. Figure l shows the nucleotide sequence of GOR47-l with the junctional sequence of lambda-gtll DNA. Sequence within the box is for GOR4'1-l and those on the left and right sides of the box are for the lambda phage.

8) Determination of the ~rimarv structure of the ~rotein coded for bv NANB heDatitis related cDNA clone (GOR~7-l) From the nucleotide sequence determined under 7) above, ORF of G0~4'7-l linked with ORF of ~-galactosidase of lambda-gtll in frame was determined as per Figure l. Lambda-gtll phage DNA used for preparation of cDNA libraries from which GOR47-l was derived is a so called expression vector and 2~ expresses fusion protein of ~-galactosidase and cDNA derived protein by assembling cDNA in operon of lacZ gene of lambda-gtll. Out of 6 possible reading frames, only one frame can fuse in frame with ORF of ~-galactosidase of lambda-gtll as shown in Figure l. There is no stop codon in this ORF
encoding 5~ amino acids. Under this invention, ~-5 ~

galactosidase can be replaced by such expression proteins as alkaline phosphatase and superoxide dismutase.

9) earch for cDNA clone overlaDDinq in seauence with NANB
heDatitis related cDNA clone (GOR47-1) From GOR47-1 phage, GOR47-1 DNA and GOR4'7-1 DNAC
were cut out as insert DNA, and from recombinant phagescript obtained, GOR47-1 RNA and GOR47-1 RNAC were prepared by T3 and T~ promoters. They were all labeled with radioisotope. When they were used as probes to screen the cDNA libraries prepared under ~) above, cDNA clone (GOR gab DNA), while overlapping with GOR47-1, had a longer sequence. The base sequence of GOR
gab DNA was determined in the same way as described above.
GOR gab DNA has the nucleotide se~uence shown in list 1. GOR 47-1 DNA corresponds to nucleic acid numbers 1703-1868 o~ GOR gab DNA.

10) GOR qab Drotein encoded bY NANB heDatitis related aene GOR qab DNA
GOR gab protein which contains 661 amino acids was obtained by search for a sequence overlapping with GOR 47-l ORF from 6 possible ORF which were coded in GOR gab DNA.
List 2 shows the amino acid sequence of GOR gab protein. The ORF coded by GOR 47-1 corresponds to amino acid numbers 569-623 in the sequence of GOR gab protein.

2 ~ 4 11) Determination of NANB heDatitis related e~itoDe (GORa) ncluded in GOR qab _ otein The following experiments were conducted using fusion proteins and synthetic peptides in order to determine NANB hepatitis re]ated epitope.

(11-1) Purification of fusion Drotein made bv NANB hePatitis related cDNA clone GOR47-1 and lambda-qtll and ~-qalactos dase E. coli Y1089 was lysogenically infected with the NANB hepatitis related gene (GOR47-1) obtained under 6) above to make lysogen. (Lysogen was prepared according to the method described in "Constructing and Screening cDNA Libraries in lambda-gtll", Thanh V. Huyuh et al., DNA Cloning Vol. 1, a practical approach, ed. by D.M. Clover, pages 949-78, IRL
Press, Oxford, 1985.) A~ter culturing and inducing expression of protein by IPTG lisopropyl-~-D-thiogalactoside), and destruction of the lysogen by means of freezing-thawing and sonication, lysate was obtained and subjected to an affinity chromatography column (Sepharose 4B Immobilized IgG Fraction Rabbit anti-~-galactosidase; Cappel, ~SA) to obtain the fusion protein.
(11-2) PreParation of svnthetic PeDtides oriqinated in GOR47-1 ORF
The following three peptides were synthesized by Merrifield's solid phase method. Numbers indicate positions in the sequence of GOR gab protein.

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spGORl (list 8) Nos. 569-597 spGOR2 (list 9) Nos. 583-609 spGOR3 Nos.597-622 (11-3) EDitoDe maD~inq bv EIA (Enzvme Immuno AssaY) and sPGORa-epi As described in the examples, antibody against fusion protein and synthetic peptides prepared in (11-1) and (11-2) above were detected by the EIA method. As Figure 2 shows, fusion protein (B-Ga1/GOR), spGORl and spGOR2 had identical antigenicity, but spGOR3 did not.
According to this result, epitope was identified as the following sequence of oligopeptide spGORa-epi (list 10) shared with fusion protein and two synthetic peptides.
GRRGQKAKSNPNRPL
Oligopeptide spGORa-epi was obtained by synthesis.

12) Another ePitoDe on GOR aab Protein, s~GORb-eDi The two most hydrophilic regions were found in GOR
gab protein provided in 10) above. One of them was spGORa-epi 2 ~

of (11-3). Another region was revealed to have NANB
hepatitis related epitope from the result of experiments concerning reactivity against antibody, when using synthetic peptide of the region, as shown in the examples.
The amino acid sequence of the synthetic oligo-peptide, spGORb-epi, was as follows:
VAKQHVRDGR KDSLDGFV (Y) 18 amino acid residues correspond to numhers 346-363 of GOR gab protein, while C-terminal (Y) was added for peptide design. Generally (Y) is tyrosine for radio-labelling, though another amino acid(s) can be utilized (e.g. cysteine) for combining with such a protein as alkaline phosphatase.

13) Preparation of anti~ody specific to NANB hepatitis related antigen Monoclonal and polyclonal antibodies were obtained by immunizing mice, guinea pigs, goats, horses, etc. with fusion proteins and synthetic peptides, GOR gab Protein, GOR
47-1 Protein, spGORl, spGOR2, spGORa-epi and spGORb-epi bearing the NANB hepatitis related epitope which were determined under 11) and 12) above.

14) Preparation of antigen assay by specific antibody to the NANB hepatitis related antigen Antibodies prepared under 13) above were labelled with FITC (fluorescein isothiocyanate) to make a probe for 2~08~4 staining sample sections, such as liver tissue of patients infected with NANB hepatitis, to locate or examine locality of antigen specific to NANB hepatitis in tissue. An assay system was also developed by labelling specific antibodies with peroxidase or biotin to detect NANB hepatitis related antigen in patient's sera or plasma. Thi.s assay employs the "sandwich method" in which microplates or beads are coated with speci~ic antibodies, and samples and labeled antibodies are applied in sequence for reaction.

15) Detection of NANB heDatitis related ~ene bY Polvmerase Chain Reaction (PCR~
According to the method described under 3) above, viral fractions were obtained by centrifugation of sample sera or plasma and subjected to PCR after treatment with SDS/Proteinase K cocktail and extraction of nucleic acid with phenol. Primer used for PCR was oligonucleotides Gl and G2, each consisting of 20 nucleotides as shown in Figure 3 and located at both ends of NANB hepatitis related cDNA GOR4'7-1.
Gl corr~sponds to 20 bases of 5' terminus of GOR47-1 DNA and is closest to 5' side of the sense strand, while G2 corresponds to 20 bases of 5' terminus of GOR47-1 DNAC and i.s closest to 5' side of anti-sense strand. As a result, the presence of PCR product of expected length (same 166 bases as 2 ~ 5 ~

GOR 47-1) was confirmed in both chimpanzee and human plasma which was suspected to be NANB hepatitis.

Examples 1~ GOR qab DNA.
The nucleotide sequence of NANB hepatitis related cDNA (GOR4'7-1) was determined in the following way.
GOR47-1 phage obtained in the above-mentioned 6) was purified, its DNA cleaved by EcoRI to obtain cDNA, that cDNA
subcloned to EcoRI site of Phagescript (Stratagene, USA) and its nucleotide sequence determined by Sanger's method.
Nucleotide sequence of the linking part of the insert arm of GOR47-1 phage DNA was similarly determined by the primer derived from the phage DNA. Those sequences revealed that GOR47-1 DNA was inserted in the lambda-gtll DNA with the nucleotide sequence and direction shown in Figure 1, and insert DNA, that is GOR4'7-1 DNA, was cut out from DNA47-1 phage DNA. Figure 1 shows the nucleotide sequence of GOR47-1 together with its linking part with lambda-gtll DNA. ~he sequence within the box is that of GOR47-1 and sequences on the right and left side of the box are those of lambda-gtll DNA.
GOR47-1 DNA has the nucleotide sequence shown in list 5.

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In the similar way, GOR47-1 DNAC (having DNA
complementary to strain GOR4'~-1 DNA) was cut out from GOR47-1 phage DNA. GOR4'7-1 DNAC has the nucleotide sequence shown in list 6.
In the next step, GOR4'7-1 RNA and GOR4'1-1 RNAC
(which have the nucleotide sequences shown in list 3 and list 4) were prepared from recombinant phage script of GOR47-L DNAC
using T3 and T7 promoters, they were labeled with radioisotope and used as probe to search the cDNA libraries prepared in 5) above, and GOR gab DNA, clone of cDNA, overlapping with but having longer sequence than GOR47-1, was obtained. The nucleotide sequence of GOR gab DNA (list 1) was determined in the same way as above.
It is well known in the art that one or more nucleotides in a DNA sequence can be replaced by other nucleotides in order to produce the same protein. The present invention also concerns such nucleotide substitutions which yield a DNA sequence which codes for GOR gab Protein (the amino acid sequence of which is described above).
2) GOR aab Protein.
From GOR gab DNA, GOR gab Protein coded by it was obtained. ~ambda-gtll phage DNA used for preparation of cDNA
libraries from which GOR gab DNA was derived is a so called "expression" vector and expresses fusion protein of ~-2 8 ~ 4 galactosidase and cDNA deriv~d protein by assembling cDNA in operon of IacZ gene of lambda-gtll. Among possible reading ~rames of GOR gab DNA, only one readin~ frame fuses in frame with open reading frame (OR~') o~ ~-galactosidase of lambda-gtll.
Amino acids sequence o~ GOR gab Protein are shown in list 2. It is well known in the art that one or more amino acids in an amino acid sequence can be replaced by equivalent other amino acids, as demonstrated by U.S. Patent No.
4,737,487 which is incorporated by reference, in order to produce an analog of the amino acid sequence. Any analogs of GOR gab Protein of the present invention involving amino acid deletions, amino acid replacements, such as replacements by other amino acids, or by isosteres (modified amino acids that bear close structural and spatial similarity to protein amino acids), amino acid additions, or isosteres additions can be utilized, so long as the sequences elicit antibodies recognizing NANB antigens.
In this invention, S-galactosidase can be substituted by such expression proteins as alkaline phosphatase and superoxide dismutase.

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3) NANB hePatitis antiqen related eDitoDe bY svnthetic pe~tide based on the amino acid seque~ce coded for bv GOR
aab DNA.
Synthetic peptides of various lengths and for various regions based on the ORF of GOR gab DNA were prepared and examined for reactivity with NANB hepatitis related antibody to determined the location of NANB hepatitis epitope.
Polystyrene microplates coated with synthetic peptide prepared by Merrifield's solid phase method as solid phase, sample sera as primary antibody, and peroxidase labeled anti-human IgG or IgM antibodies as secondary antibody for color reaction were used.
As a result, spGORa-epi and spGORb-epi ~ere determined as NANB hepatitis related epitopes. SpGORa-epi has the amino acid sequence shown in list 10 and spGORb-epi shown in list 1l.

4) NANB he~atitis antibody detection sYStem usinq ~roteins and DeDtides described above as antiqen.
An example of experiments of NANB hepatitis antibody detection system (Enzyme Immuno Assay) using GOR gab protein and peptides is shown below.
50 ~1 each of GOR gab Protein or peptides having 10 or more amino acids residues dissolved in Tris-HCl buffer (lOm~, pH 7.5) and adjusted to 5 ~g/ml concentration was dispensed in each well on Costar vinyl plates (Toyobo, Japan~

and incubated overnight at room temperature (coating of peptide). After washing the wells tO.1% Tween 20, lSOmM NaCl;
all washing procedures hereafter in this e~periment, unless otherwise stated, were with this solution), 100 ~1 each o~ the blocking solution (0.1~ Tween 20, 150mM NaCl, 30% fetal calf serum) was dispensed in the wells for overnight incubation at 4C on a vibration free. After washing the wells, 50~1 each of sample plasma, or serum preliminary diluted 30 times with the above-mentioned blocking solution, was dispensed in the wells for incubation at room temperature for 30 minutes on a vibrator. After washing the wells, 50~1 each of peroxidase labeled anti-human IgG or IgM mouse monoclonal antibody solution was dispensed in the wells for incubation at room temperature for 30 minutes on a vibrator, then peroxidase substrate solution was added for color development and absorbance measurement at wavelength 492nm on a spectrophotometer.
(1) When samples from 100 chronic liver disease patients (mixture of hepatitis B and NANB hepatitis) were assayed by the EIA method, frequency by the assay using the protein or peptides under this invention turned out to be high for NANB chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, while low ~or chronic liver disease, liver cirrhosis and hepatocellular carcinoma caused by hepatitis B
virus, lupoid hepatitis of which autoimmune disorder was :

2 ~ 4 suspected, and primary biliary cirrhosis. The assay also showed low frequency for samples from normal subjects, thus proving the efficiency of the protein and peptides under the present invention for use in detection system of NANB
hepatitis related antibody.

(2) As Figure 4 shows, GORa antibody frequency detected by EIA using spGOR2 was as high as '16~, 56% and 56%
for NANB related chronic hepatitis, liver cirrhosis and hepatoma respectively, while it was as low as 2%, '7% and 0%
for hepatitis B related chronic hepatitis, liver cirrhosis and hepatoma respectively. It was 0% and 0% for lupoid hepatitis and primary biliary cirrhosis which were supposed to be closely associated with autoimmune mechanism, or was only 2 (1%) out of 200 for normal subjects. This proved a close relation of GORa antibody with NANB hepatitis.

(3) Change of GORa antibody in an acute NAN~
hepatitis patient.
GORa antibody positive frequency turned out very hiqh for chronic hepatitis, liver cirrhosis and hepatoma as shown above, and the antibody appeared in very early period for acute hepatitis. A typical example of an outbreak of acute hepatitis is shown in figure 5 (needle stick accident in 2 ~

which a nurse stuck herself with an injection needle while drawing blood from a patient with NANB chronic hepatitis).
In this case, both Ig~ and IgG classes of GORa antibodies were detected on the 49th day a~ter the needle stick accident while HCV antibody was detected (Ortho HCV Ab ELISA test; Ortho Diagnostic Systems, Japan) as late as on the 131st day after the accident. This has proven that GORa antibody is effective in early detection of infection.
GORa antibody could be derived from GOR 47-1 protein, spGORl, spGOR2, and spGORa-epi.

5) Detection of NANB heDatitis related antibodY bY the fusion Drotein containina NANB eDitoDe sDGORa-epi as antiaen While the fusion protein of GOR 47-1 and lambda-gtll-~-galactosidase was used, ~-galactosidase can be substituted by such expression proteins as alkaline phosphatase and superoxide dismutase.
The fusion protein obtained above was electrophoresed by SDS-PAGE and its pattern was transferred onto nitrocellulose membrane by the Western blotting technique. Sample plasma was applied onto this membrane and after incubation anti-human immunoglobulin labeled with peroxidase was added for detection of the antibody through color reaction.

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The Western blotting method was used for detection system as follows: The nitrocellulose membrane with the electrophoresed pattern transferred onto it was rinsed for 10 minutes, dried natural:ly, shredded into strips 3.5 mm wide, and those strips were immersed in TBS (50mM Tris-Cl buffer, pH
7.4, 150 mM NaCl) containing 2% skim milk at room temperature for 1 hour or at 4C overnight (blocking), then washed with TBST (TBS containing 0.0S% Tween 20). After immersion at room temperature for 1 hour or at 4C overnight in the primary antibody diluted 30 times with 3% BSA (primary antigen-antibody reaction), those strips were washed with TBST, immersed at room temperature for 30 minutes in biotinylated anti-human IgG or anti-human IgM (Vectastain ABC kit; Vector Laboratories Inc., USA) diluted with TBS (secondary antigen-antibody reaction), washed with TBST, then immersed in peroxidase labeled mixture of biotin and avidin (Vectastain ABC kit; Vector Laboratories Inc., USA) at room temperature for 30 minutes (biotin-avidin reaction), then washed with TBST
followed finally by addition of coloring reagent to examine the presence of reactive substances. The coloring reagent was prepared by adding 25 mg of DAB (3,3'-Diaminobenzidine tetrahydrochloride; Sigma, USA) to 50 ml of 50 ~M NaPO~ pHrl ~ 4 and, after dissolution of DAB, 1 ml of 5% CoC13-6HaO, 5%
(NH4)2Ni(SO~)2 6H20, then, 50 ~1 of 30% H202. When antibody was positive, a band of about 120 K Dalton was stained dark blue.
- ~4 -2 ~ 3 (1) Detectability of NANB hepatitis related antibody (GORa antibody) in patients with chronic liver diseases.
Sera of lOO patients with chronic liver diseases (40 cases of hepatitis B and 60 cases of non-B hepatitis) were assayed by the Western blot analysis. Results are shown in Tables 1 and 2, and Figure 6. As shown in Tables 1 and 2, among 100 case~, GORa antibody was positive for 52 cases in total and its rate was much higher for the sera of non-B
hepatitis cases (80%) than for the sera of hepatitis B cases (10%). Especially in chronic non-~ hepatitis cases, GORa antibody was positive for 27 cases out of 30 cases (90~).
When GORa antibody was compared with Chiron's HCV antibody (Ortho ~CV Ab ELISA test; Ortho Diagnostic Systems, Tokyo, Japan), the former showed lower positive rate for the sera of hepatitis B cases (10%, 13%) and conversely higher positive rate for sera of NANB hepatitis cases (80%, 68%) than the latter. Furthermore, as shown in Figure 6, there were 14 NANB
hepatitis cases which GORa antibody detected but HCV antibody failed to detect, while there were only 6 converse cases.

l2) GORa antibody in sera from donors having abnormal liver function.
Frequency of GORa antibody and HCV antibody for sera of randomly selected 42 donors (ALT over 80, hepatitis B
negative) were, as shown in Table 3, 20% and 17~ respectively.

2~8~

Sera were tested in the method described in 1) immediate~y above. In five cases, both GORa antibody and HCV antibody were positive. ALT (alanine aminotransferase) level in serum is one of the markers which indicate liver function and is high (abnormal) when the host is infected with viral hepatitis, but it is also high when liver is malfunctioning due to some other causes such as alcohol, drugs, obesity, etc.
The group consisting of 42 donors was consciously healthy but was unconsciously high in ALT level and differed from the groups in ~1) above in that the cause of the high ALT level was not checked in this group while that of the groups in ~l) was chec~ed (chronic hepatitis, liver cirrhosis, or hepatoma).
The difference in frequency of GORa antibody between this group and those in (1) may be attributed to this fact.

(3) Comparison of EIA for the detection of G~Ra antibody using synthetic peptide and Western blot analysis using protein fused with ~-galactosidase.
100 sera from patients with chronic type-~ or type-NANB liver diseases were tested by EIA for GORa antibody and most of them turned out to be positive by both EIA and Western blot analysis, but 11 samples were positive by EIA but negative or weakly positive by Western blot analysis, suggesting that the former detects GORa antibody at higher sensitivity than the latter (Table 5).

2~8~4 6) The antibodv detection svstem usinq sDGORb-e~i which contains NANB hePatitis related eDitoPe of the invention, GORb, as antiqen was Prevared in the same method as in examDle 1~.
The results shows the efficiency of spGORb-epi for detection of NANB hepatitis as follows; GORb antibody was detected in 8 out of 290 cases ~3%) in normal subjects, 1 out of 36 cases (~%) in type-B liver disease and 17 out of 54 cases ~31%) in NANB hepatitis as shown in Fig 7 (cutoff value was OD~92 = 0-4-7) PreDaration of monoclonal and Dolvclonal antibodies.
Speci$ic monoclonal and polyclonal antibodies were obtained by immunizing such animals as mice, guinea pigs, rabbits, goats and horses with GOR gab Protein and the synthetic peptides bearing NANB hepatitis antigenic epitope described in the above example.

8) Antiaen detection assa~ usina antibodv sDecific to NANB
heDatitis related antiaen.
By staining tissue sections of liver, etc. from NANB
hepatitis patients with the aforementioned speci~ic antibody labeled with FITC as probe, the presence and locality of NANB
hepatitis specific antigen in tissue was examined. By labeling the specitic antibody with peroxidase or biotin, assay system for detection of NANB related antigen in patient 2 ~

serum or plasma was de~eloped. The system was the sandwich method in which polystyrene microplates or beads coated with NAN~ hepatitis specific antibody were used as solid phase and after addition of samples for reaction with the solid phase, labeled specific antibody was added for the second reaction.
Experiments of those assays confirmed the effectiveness of monoclonal and polyclonal antibodies under the present invention for detection of NANB hepatitis related antigen.

9) Detection of NANB he~atitis related aene bv Pol~merase Chain Reaction (PCR) According to the method described under 3) above, viral fractions were obtained by centrifugation of sample sera or plasma and subjected to PCR after treatment with SDS/Proteinase K cocktail and extraction of nucleic acid with phenol. Primer used for PCR was oligonucleotide Gl and G2, each consisting of 20 nucleotides as shown in Figure 3 and located at both ends of NAN~ hepatitis related cDNA GOR4'7-1.
Gl corresponds to 20 bases of 5' terminus of GOR4'7-1 DNA and is closest to 5' side of the sense strand, while G2 corresponds to 20 bases of 5' terminus of GOR4'7-1 DNAC and is closest to 5' side of anti-sense strand. After addition of primer G2 (antisense) to the above mentioned nucleic acid 2~ fractions as the primary reaction in PCR, 4 nucleotides and 2 ~

transcriptase were added for reaction and synthesis of primary cDNA. Primer Gl was then added and the standard PCR reaction was followed for 36 cycles using Taq polymerase and the automatic temperature control unit tCetus Corp., U.S.). The product (DNA) thus produced was separated by agarose gel electrophoresis and the presence of an expected length (same 166 bases as GOR47-1) of DNA fragment (identified by * in Figure 8) was confirmed. Chimpanzee (CH19) plasma ~rom which NAN~ hepatitis related cDNA clone GOR47-1 was derived contained PCR product of 166 bases (Figure 8, Lane 1). A
similar band was separated from the plasma of chimpanzee (CH413) having known infectious unit of 10~ CIUJml (chimpanzee infectious units/ml) (Figure 8, Lane 2). Human plasma (T.S) known to have 106 CIU/ml also showed the similar length of band but its a~ount was about lJ10 of that of the aforementioned chimpanzee having 107 CIUJml. Patterns on the extreme left of the figure are those of molecular markers.

10) Seroconversion of GORa antibodv in acute NAN~ hevatitis patisnt Table 4 contains data from a nurse who had been accidentally exposed to a needle stick contaminated with blood from a non-A, non-B hepatitis patient resulting in an acute hepatitis. As the table shows, there was no GORa antibody before infection and it was detected only after infection. At - 2~ -2 ~ 8 ~ ~

the initial stage of infection, GORa antibody of the IgM class was also positiYe. Emergence of HCV antibody, on the other hand, was very late. At the 8th week after infection, GORa antibody was already positive, while Chiron's HCV antibody was negative (Ortho HCV Ab ELISA test; Ortho Diagnostic Systems, Tokyo, Japan) until the 1'7th week after infection; this proves that, at least for this case, serological diagnosis of NANB
hepatitis infection at an early stage is possible only with GORa antibody. Expression of IgM class GORa antibody in the acute phase presents another important significance. It is not easy to discern between an acute exacerbation of chronic hepatitis and acute hepatitis. They can, however, be distinguished by testing for GORa antibody IgM class and accurate diagnosis becomes possible.
The antigens and antibodies of the invention provide diagnostic reagents capable of quicker-and wider range of diagnosis of NANB hepatitis than any other conventional reagent, and can be used at blood centers, blood derivatives manufacturers, transfusion departments of hospitals and many other places for elimination of blood carrying NANB hepatitis agent from transfusion blood and blood derivatives. Screening tests by the diagnostic reagents using the antigen and antibodies under this invention will provide op~imum means for prevention of post transfusion hepatitis which has long been a grave concern.
The nucleic acids and protein under this invention are manufactured not only as important reagents for serological and virological study of NANB hepatitis causative agent and for pathological study of the disease caused by it, but also as indispensable materials for manufacture of the aforementioned antigens and antibodies.
U.S. Patent Application Serial No. 0'7/5~0,604, filed on June 19, 1990, is hereby incorporated by reference for its teachings that the materials of the present invention may be utilized in detection kits and as vaccines.
Further variations and modifications of the invention will become apparent to those skilled in the art from the foregoing and are intended to be encompassed by the claims appended hereto.
Japanese Priority Application 104010/91, fi:Led on February 8, 1991, is relied on and incorporated by reference.

Table 1: GORa ~ntibody detected in patienLs with chronic liver disease ~ 8 ~ ~
. .
*

No. Patient Diagnosis GORaAntibody HCV Antibody A.O. (M)Chronic hepati~is/B
2 M.O. (M) 3 S.F. (M) "
Y.l. (~1) ' + +
H.N. (M) "
6 T.O. (M) "
7 S.Y. (F) 8 K.G. (M) "
9 A.l. (hl) "
K.N. (M) "
Il H.K. (M) "
12 N.T. (~vl) " -13 Y.T. (M) "
14 T.K. (M) "
K.A. (M) "
16 K.S. (M) " + +
17 T.N. (F) "
18 H.T. (M) "
19 K.G. (F) "
T.T. (M) "
2 I T.S. (M) "
22 H.A. (M) "
23 S.T. (M) "

24 S.O. (M) "
M.W. (M) "
26 T.S. (M) "
27 T.S. (M) " +
28 F.N. (M) Chronic hepatitis,~on-B + +
29 G.O. (M) " + +
M.O. (M) , +
3 I R.F. (M) ' + +
32 S.K. (F) " +
33 Y M. (M) +
34 T.Y. (F) " - +
T.Y. (M) " + +
36 Y.K. (M) "
37 K.l. (M) " + +
38 K.O. (F) " + +
39 H.S. (M) " + +
A.H. (F) " + +
4I K.l. (M) " + +
42 K.G. (M) 43 Y.K. (M) +
44 M.S. (M) " + +
C.A. (F) " +
46 M.Y. (F) " +
47 K.l. (F) " + +
48 R.N. (M) " +
49 F.F. (F) " +
T.hl. (1~/1) " +

- :32 -51 H.K. (F) " +
52 H.S. (F) " + +
53 Y.K. (M) ~ " - +
54 T. I. (F) " + +
S. ?. (F) Chronic hepati~is/Non-B + +
56 H. M . (M) " +
57 S.A. (F) " +

58 Y.M. (M)LiverCi~hosis~B - +
59 N.Y. (M) "
60 H.l. (M) " - -6I S.K. (M) "
62 M.K. (M) "
63 H.F. (F) ' - -64 S.A. (hl) Y.F. (h~l) " - -66 T.F. (F) " - -67 K.K. (F) "
68 T.K. (M) " + +
.
69 M.N. (M)Liver Cirrhosis/Non-B - +
K.O. (F) 71 K.M. (M) " + +
72 F.K. (F) + +
73 H.S. (F) " + +
74 H.O. (F) " + +
T.l. (M) " ~ +
76 Y.A. (F) " +
77 TK. (M) " +
78 S F. (M) " - +
79 Y.I. (M) +
H.H. (F) " +
8I R.S. (hl) " + +
82 ?.N. (M) " + +
83 K.K. (M) " - +
84 K.Y. (M)Liver Cirrhosis~Non-B +
K.T. (F) " + +
86 G.N. (M) " + -~

87 T.S. (hl)Hepatom~JE~ - -88 N.T. (M) "
-89 T.Y. (M)Hepatoma~on-B
M.I. (F) " + +
9 I R.A. (M) " ~ +
92 J.S- (M) ~
93 K.S. (M) " +
94 S.K. (M) " +
T.T. (M) 96 S.A. (M) " f +
97 H.A. (M) "

98 U.K. (F)HepatomalB + +
99 ?.O. (M) 100 H.O. (~
Ren~arks:
I) "~" and "Non-B" in the "Diagnosis" column denote patients clearly associated with hepatitis B virus and those not associated with the virus respectively.
2) i column shows result of assays by Onho HCV Ab ELISA test kit for comparison purpose.
3) (M) and (F) in "Patient" column designate male and female respectively.

- :34 -Table 2: Frequency of NANB GOR47- 1 antibody in patients with liver diseases 2 ~ 6 ~ ~ ~ 4 - Number ofFrequency of *
Disease Group Patient GORa Antibody HCV Antibody Number (Rate) Chronic hepatitis/B 27 3 (11%) 2 ( 7%) Chronic hepatitis/Non-B 30 27 (90%) 21 (70%) -Liver cirrhosis/B 11 1 ( 9%) 3 (27%) Liver cirrhosis/Non-B18 14 (78%) 14 (78%) Hepatom~B 2 0 ( 0%) 0 ( 0%) Hepa~oma/Non-B 12 7 (58%) 6 (50%) B Total 40 4 ( t 0%)5 ( 13%) Non-B Total 60 48 (80%) 41 (68%) Grand Total 100 52 (52%) 46 (46%) -Remarks:
~ colllmn shows for comparison nun-ber (rate~ of samples teslcd positive for HCV in Table 1.

- 3~ --T~ble 3: GORa antibody detected in donors with malfunctioning liver.
Donor GORa * Donor(:;ORa *
No. Antibody HCV Antibody No. Antibody HCV Antibody 2 - - 23 + +
3 - - ?.4 +
4 - 25 - +

9 - - 30 + +

I I 32 + +

+ 36 +

17 - - 38 + +

- - 41 N.T.
2 1 + + 42 +
Remarks: -I) For comparison purpose, * column shows the result of samples tested for Chiron's HCV antibody.
2) All samples were tested negative for hepatitis B virus antigen. As demonstrated in Table 2, 90% of Non-B chronic hepatitis samples were tested positive for GORa antibody, while only 20% of those "consciously healthy" population were positive for the antibody since such liver malfunction derives not only from the virus but from obesity and alcohol.
3) "N.T." means "not tested".

- ~6 T~ble 4: Seroc~nversion of GORa in an acute non-A, non-B hepatitis pa~ient ~ ~s 5; ~ 8~ 1 -GORa Antibody *
Time (Week) ALT (HCV Antibody) IgM IgG

4 32 N.T. N.T.
7 889 N.T. N.T.
8 381 + +
Il S + +
14 20 N.T. N.T.
17 41 +
19 IS N.T. N.T. +
21 77 N.T. N.T. +
22 3~ + - +
27 18 + - +
250 N.T. _ N.T. N.T.
Remarks:
I) Above table shows a case of a nurse infected with hepatitis by needle prick accident.
Time indicates number of week(s) elapsed after the accident. The patient, the source of the blood collected, was also tested positive for GORa antibody.
2) Result of the tests for Chiron's HCV ~re shown in * column for comparison.
3) N.T. means "not tested".

T~b!e S Comparison of E~IA using spGOR2 ant.ibody and Western blot analysis using 2 ~ ~ O ~ 4 No. EIA West. No. EIA West. No. EIA West. No. EIA West.
28 - 26>2000 + 511603 + 76 - 53 2 57 - 2771 - 5253 - 77>2000 3 107 ' - 2863 - 5355 - 78>2000 4 >~000 + 291557 + 54155~ + 79>2000 5474 - 3037 - 5552 i 80>2000 650 - 3134 - 561512 + 81 386 7134 - 3~87 - 57910 - 82>2000 8>2000 + 33>2000 + 58 >2000 + 83 1554 1051 - 3563 - 60110 _ 85>2000 11807 _ 3661 - 6129 - 86 49 1262 - 3732 - 621941 + 87 75 13101 - 381039 + 6355 - 881630 1499 - 3930 - 64789 + 89 33 151886 + 401731 + 65 >200Q + 90 1283 16 ~2QOQ+ 4149 - 661945 + 91>2000 1777 - 42265 + 6753 - 92 35 18>2000 + 43>2000 + 68200 - 93 64 19397 _ 4451 - 6939 - 94>200Q

21>2000 - 4634 - 71167 - 96>2000 22>2000 + 47>2000 + 721582 + 97 69 23>2000 + 48>2000 + 73469 - 98 729 24 >2000+ 4987 - 74 >2000 + 99 >2000 25 68 - 50?2QOO + 75 607 + 100807 Remarks: "West." indicates Western blot analysis $'~

.., . .. .. ..., . ... .., . ~, ., , ~, . ..

SeLIuenc~ t Sequence lis~ 1. GOR ~ab DNA
GAATI'CCCCC TGCCCTCTGA TCAGCCTGCC CAGAGCTTCG GGCTCCGGGT 50 : TGCACAGATG CCAAGAGAAC CCGGGTGGCC AGCAGCAGCC AACGCTCCAG 350 ; TGGCTCCAAG GTCGGCAGAC AGCCAGGGAA GACGCGCAAC AGGTCAGGGA 400 TG~CCTTGGC CCCAGGCCCC TTCCACAACC GCCATCAGTC CCGAGAGCTC 1700 Seql~ence list 2. GOR gab protein .EFPLPSHQPA QSFGLRVPQM HNQASAFVDI QAEPQNRGPA VPPACLKMVT 50 DYLGQ~IQDS QDGHNSSEDA NACLQLVMWK VRQRAQIQPR HRSASPAALA 550 SAHPRPLSLH H 6~1 Sequence list 3. GOR47- 1 RNA

: AGAGGGCAAA AAGCCAAGAG UAACCCCAAC CGGCCACUCC CAGUCCCCCG 100 Sequence list 4. GOR47-1 RNAC

Sequence list 5. GOR47-1 DNA

Sequence list 6. GOR47-1 DNAC

Sequence list 7. GOR47- I protein CPPRR~AKET GAVDGRRGQK AKSNPNRPLP VPRNPCRGPS GLSPSLCPSQ 50 Sequence list 8. spGORl CPP~RKAKET GAVDGRRGQK AKSNPNRPL 29 Sequence list 9. spGOR2 Sequence liStlO. spGORa-epi Sequence list 11. spGORb-epi

Claims (6)

1. An oligopeptide spGORb-epi comprising the following amino acid sequence:
V A K Q H V R D G R K D S L D G F V (Y) or analogs thereof.

2. The oligopeptide according to claim 1, wherein said amino acid sequence comprises V A K Q H V R D G R K D S L D G F V.

3. A non-A, non-B hepatitis antibody detection reagent comprising the amino acid sequence according to claim 1 as antigen.

4. A non-A, non-B hepatitis related antibody detection reagent comprising a fusion protein of the amino acid sequence according to claim 1 and a conventional protein for its expression.

5. A non-A, non-B hepatitis specific monoclonal or polyclonal antibody reactive with an antigen comprising the amino acid sequence according to claim 1.

6. A non-A, non-B hepatitis related antigen detection reagent comprising the specific antibodies according to claim 5.